Y.-L. Chen et al.
Journal of Inorganic Biochemistry 180 (2018) 194–203
2.1. Materials
2.4. Electrochemical measurements
PIH was purchased from Santa Cruz Biotechnology, Heidelberg,
Cyclic voltammetry (CV) measurements were performed with an CS-
120 device (Corrtest). Solutions of SIH or PIH (2 mM) with iron(III)
(1 mM) at pH 7.42 (0.1 M 3-morpholinopropane-1-sulfonic acid, MOPS,
20% DMSO V/V) were investigated. All measurements were conducted
under N in a jacketed, one-compartment cell with a platinum disk
2
2
2.2. Preparation of SIH
working electrode (geometric area: 0.07 cm ) (Corrtest), a platinum
wire counter electrode (Corrtest) and a Ag/AgCl reference electrode.
The working electrode surface was polished, sonicated in water, and
This synthesis utilised the method reported by Hassan et al. [31].
Isoniazide (4.11 g, 30 mmol) was dissolved in 150 mL ethanol at 60 °C,
then salicylaldehyde (4.03 g, 33 mmol) was added and the reaction
mixture was refluxed for 4 h. The solution was cooled to 0 °C and a
yellow precipitate was obtained by filtration. The crude product was
recrystallized from ethanol to give SIH (6.0 g, yield 83%) as pale yellow
crystals.
air-dried immediately before use. The sweep rate was 100 mV/s. O
removed from the electrolyte solution by bubbling N through the
solvent for several minutes prior to measurement. A N atmosphere was
continuously maintained above the solution while the experiments
were in progress. The temperature was controlled by using a double-
jacketed cell and a thermostat with water bath at 25 °C.
2
was
2
2
N′-(2-hydroxybenzylidene)isonicotinohydrazide (SIH): pale yellow
solid; m.p. 247.8–248.7 °C; IR (KBr): νmax = 3438, 3203, 3006, 1684,
2.5. Kinetic spectrophotometric measurements
−
1 1
1614, 1568, 1291, 772 cm ; H NMR (400 MHz, DMSO‑d
6
) δ(ppm):
12.28 (s, 1H, NH), 11.06 (s, 1H, OH), 8.79 (d, 2H, J = 6.0 Hz, pyridine-
All kinetic studies were undertaken at pH 7.4 (25 mM MOPS) and
25 °C. The iron concentration adopted throughout was 10 μM and the
corresponding ligand concentration was chosen based on its dentate
number, 30 μM for deferiprone, 20 μM for SIH and PIH. Iron(II) sul-
phate was used as a source of iron(II) and the solutions were prepared
immediately before use. Iron(III) nitrate was used as a source of iron
(III), again the solutions being prepared immediately before use. The
usual practice of using iron(III) nitrilotriacetic acid (NTA) solutions was
not adopted because NTA interferes with kinetics of iron substitution.
The glutathione concentration was 2 mM. The UV–Visible spectra were
recorded by an HP 8453 UV–Visible spectrophotometer with a 50 mm
Hellem quartz flow cuvette being circulated by a Gilson Mini-plus #3
pump – speed capability (20 mL/min), controlled by in-house computer
software.
H), 8.67 (s, 1H, N]CH), 7.84 (dd, 2H, J = 2.0, 4.8 Hz, pyridine-H),
7
6
1
1
4
.60 (dd, 1H, J = 1.6, 7.6 Hz, AreH), 7.33–7.29 (m, 1H, AreH),
1
3
.95–6.90 (m, 2H, AreH); C NMR (100 MHz, DMSO‑d
60.9, 157.1, 149.9, 148.9, 139.6, 131.3, 129.1, 121.2, 119.1, 118.3,
16.2; MS (ESI): m/z (%) = 240; C13 , requires C, 64.72%; H,
6
) δ(ppm):
11 3 2
H N O
.60% and N, 17.41%; found, C, 64.72%; H, 4.52% and N, 17.47%.
2.3. pKa and iron stability constants
The automated titration system used in this study comprised of an
autoburette (Metrohm 765 Dosimat autoburette) and Mettler Toledo
MP230 pH meter with Metrohm pH electrode (6.0133.100) and a re-
ference electrode (6.0733.100). 0.1 M KCl electrolyte solution was used
to maintain the ionic strength. The temperature of the test solutions was
maintained in a thermostatic jacketed titration vessel at 25 °C ± 0.1 °C
by using a Techne TE-8J temperature controller. The solution under
investigation was stirred vigorously throughout titrations. A Gilson
Mini-plus#3 pump with speed capability (20 ml/min) was used to cir-
culate the test solution through a Hellem quartz flow cuvette. For the
stability constant determinations, a 50 mm path length cuvette was
used, and for pKa determinations, a cuvette path length of 10 mm was
used. The flow cuvette was mounted on an HP 8453 UV–Visible spec-
trophotometer. All instruments were interfaced to a computer and
controlled by a Visual Basic program. Automatic titration and spectral
scans adopted the following strategy: the pH of a solution was increased
by 0.1 pH unit by the addition of KOH from the autoburette; when pH
readings varied by < 0.001 pH unit over a 30 s period, they were
judged to be stable and the spectrum of the solution was recorded. The
cycle was repeated automatically until the defined end point pH value
was achieved. All the titration data were analyzed with the pHab pro-
gram [32]. Species plots were calculated using the HYSS program [33].
3. Results
3.1. Determination of pKa values
PIH was found to be relatively stable over the pH range 1–10, in
contrast to SIH which was found to be unstable at pH values < 2 (Fig.
S1 Supplementary data). This is likely to result from the hydrolysis of
the Schiff base. Thus exposure of this ligand to pH values < 2.0 was
avoided during titration studies.
The spectrophotometric analysis of the titrations of PIH and SIH
(Fig. 1) were fitted to four and three pKa values respectively. The
corresponding pKa values are given in Table 1. There is good agreement
with the study of Richardson et al. [35] and although we agree with
their assignment of pKa values for SIH, as indicated in Fig. 2, we do not
agree with the previous assignment of values for PIH. Based on studies
by Metzler and Snell [ 36], Richardson assigned their measured pKa
value of 4.20 to the phenolic function [35]. However, titration of 3-
hydroxypyridine and its analogue, pyridoxine, follows the sequence
indicated in Eq. (4) [ 37,38]. Based on these studies, we propose the
pKa assignments for PIH are as indicated in Fig. 2. These assignments
are close to those calculated for PIH by Marvin [39]. The influence of
pH on the speciation plots of the two ligands is presented in Fig. 3. With
both compounds, there is a mixture of the non-charged species and the
monoanionic species at pH 7.4.
The values of logK
termined by use of the HYSS program [33]. Analytical grade reagent
materials were used in the preparation of all solutions.
1
2
and logβ , as defined in Eqs. (1)–(3), were de-
3
+
In addition to logK
1
and logβ
2
values, pFe
values have been
adopted by chelation chemists as a convenient means of comparing the
iron(III) chelating capability of different ligands at pH 7.4. At this pH,
there can be appreciable competition for the ligand by protons. In
contrast, the logK
equilibrium constant in the absence of proton competition (Eqs. (1) and
2)).
pFe3+ values are determined from logβ values, pKa values, pH and
1
and logβ
2
values are based on the values for the
3.2. Determination of the iron(III) affinity constants
(
PIH and iron(III) were titrated in a 2:1 M ratio against KOH over the
pH range 1.3–7.5. There is an isosbestic point at 475 nm with the λ max
shifting from 473 to 465 nm (Fig. S2, Supplementary data). The data is
consistent with the formation of two complexes, namely Fe·(PIH) and
III
total concentrations of Fe and ligand. Typically, when comparing iron
(
III) chelators of clinical interest, the following conditions are adopted;
3
+
−6
total = 10
−5
[
Fe
]
M; [ligand]total = 10
M and pH = 7.4 [34].
Fe·(PIH)
2
. The affinity constants logK
1
2
and logβ were determined as
195